DESCRIPTION: (Applicant's Description) This proposal seeks to develop a novel screening system to support cancer diagnosis, tumor staging, prognostication based on the measurement of gene expression at the RNA level. The underlying hypothesis hat tumors progress because of accumulation of genetic changes, and that the genetic changes manifest themselves in altered and measurable expression of one or several genes. Current techniques limit the measurement of RNA expression levels to a few genes per experiment. Sensitive tumor cell detection and prognostication will require methods that simultaneously profile the RNA expression levels of five or more suspected oncogenes/tumor suppressor genes. Furthermore, if an oncogene is overexpressed, then its gem dosage should be determined as well. The quantitative analysis of several marker genes is crucial for sensitive tumor cell detection and accurate cell classification as well as the development of novel anti-tumor strategies. This information should help discriminate benign vs. malignant neoplasms and define prognostic markers. We will develop multicolor fluorescence in situ hybridization (FISH) in conjunction with Spectral Ima (SIm) to perform these measurements. Existing SIm instrumentation can record fluorescence spectra from nm to 1100 nm with about 10 nm resolution. This allows the unique labeling and detection of eDNA probe with commercially available fluorochromes. SIm technology can be modified to investigate the correlation cancer gene expression with tumor progression. Spatial co-localization and spectral overlap will be add by software processing of digitally recorded images, termed "spectral un-mixing" (SUN). Using artificial mixtures of cells from existing thyroid tumor and breast cancer cell lines, we will develop the software modules needed to measure intracellular levels of multiple RNA species in the same cell and to determine the assay sensitivity, accuracy, and reproducibility. The work will be extended to thyroid and breast tumor tissues increasing the number of hybridization targets and correlating gene rearrangements/amplifications with gene expression. This technology, when applied biopsy tissue from fine needle aspirates, could help diagnose and predict the course of suspicious cells in a rapid, inexpensive, and minimally invasive manner.